Molecular Characterization of a Carbamoyl-Phosphate Synthetase II (CPS II) Gene from Trypanosoma cruzi

Abstract

A number of protozoan and helminth parasites are incapable of synthesizing purines de novo and rely on salvaging preformed purines. In contrast, few organisms lack de novo pyrimidine synthesis, the pathway of which is subjected to an exquisite regulation through inhibition and stimulation of carbamoyl-phosphate synthetase II (CPS II) activity. Previous works in this laboratory (Aoki, 1994) showed that, in Schistosoma mansoni and Ascaris suum, as well as in mammals, CPS II occurs as the first enzyme in a multifunctional protein with the second and third enzymes of the pathway, aspartate carbamoyltransferase (ACT) and dihydroorotase. However, our attempt to purify these enzymes from trypanosomatids resulted in the separation of CPS II and ACT activities, suggesting the enzymes to be independent proteins (Aoki and Oya, 1987a). The partially purified CPS II showed kinetic and regulatory properties different from those of prokaryotic and eukaryotic CPSs II. To gain an insight into these biochemical differences, we have partially determined the sequence of CPS II cDNA from Trypanosoma cruzi, a protozoan flagellate that causes Chagas’ disease in man in Latin America, and the deduced amino acid sequence has been characterized. We report here that trans -splicing may bring about the maturation of pre-mRNA and that a short Polypeptide links the glutaminase and carbamoyl-P synthetase components, homologues of the light and heavy subunits of Escherichia coli CPS II.